OBJECTIVES: We plan to study the mechanism of B12 absorption by: a) isolating and purifying the intrinsic factor receptor protein from the ileal mucosa of dog; b) isolating and purifying intrinsic factor from the dog gastric mucosa; c) determining the amino acid and carbohydrate structures of these proteins and determining the critical involvement of each moiety in the interaction of IF with B12 and in the interaction of IF-B12 with the receptor protein; and d) studying the transmembrane flux of IF-B12 from the surface of mucosa through the epithelial cell using cell culture and immunochemical methods. We plan to purify analytical amounts of B12-binding protein TC-I and TC-II in order to: a) study the sites and kinetics for the synthesis and degradation of these binders using immunochemical methods; and b) develop radioimmunoassays which could measure precursor, and degradation forms of these proteins which do not bind B12. We plan to solubilize and characterize the TC-II receptor protein from cell membranes. We plan to develop antiserums to specific B12 analogues and use these antiserums to study the intracellular localization of B12 cofactors by immunofluorescence, and develop specific radioimmunoassays for these analogues in blood and tisues.